Gel electrophoresis (“GE”) is a commonly used electrophoretic separation technique for the analysis of proteins and nucleic acids. GE uses a polymer-based sieving matrix composed of a polyacrylamide gel (e.g., for separation of proteins) or an agarose gel (e.g., for separation of nucleic acids, such as DNA or RNA). Although GE is a mature technique, with several thousands of publications since the 1950s, the field has remained a subject of intense investigation [1]. Indeed, many disadvantages of the current polymer-based sieving matrix approaches for GE have been identified and include lack of repeatability in the production/polymerization of the sieving matrix, single use, and inability to reproduce the same result for similar experiments. Also, the concentration, cross-link density, and rate of formation of the gel can further affect the optical properties, rigidity, and pore size [2]. Other causes of concern with current GE techniques include Joule heating, non-uniform polymerization [4], adhesion of the gel to the support [3] and structural inhomogeneities [5, 6]. Finally, the polymerization of the components used to produce the gels can be significantly affected by slight changes in humidity, temperature, monomer and cross-linker concentration and/or mixture ratio, and personnel performing the process (i.e., technician dependent). As a result, significant variations often exist between experiments performed by the same individual at different times or performed by different individuals in different laboratories. Accordingly, experimental reproducibility is a major problem with current GE techniques and significantly influences the results when repeated experiments are needed to verify a biological research outcome.
Despite the inherent limitations in current GE techniques, however, separation science has continued to play a vital role in chemical and biomedical analyses, and remains indispensable in a wide variety of fields including proteomics, genetics, clinical diagnostics, environmental protection and forensics. The ever increasing need for advancements in proteomics and genetics for combating disease and furthering the understanding of biological systems has thus necessitated the need for superior separation tools. Therefore, researchers have begun to investigate the feasibility of microfabrication for the separation of one or more molecules in a particular sample.
To date, however, only a few investigators have addressed the need to develop alternative GE techniques based on microfabrication methodologies. For example, some researchers, who recognized the limitations of current macroscale polyacrylamide gel electrophoresis (PAGE) techniques, have developed a microfabricated electrophoretic device to study the sieving effect in protein electrophoresis [4]. Such a device included a fluidic channel filled with an “artificial gel,” which consisted of a series of staggered posts approximately 2 micrometers in diameter, with the post pitch determining the pore size of the “gel.” Proteins were observed to migrate through the sieving matrix in this artificial gel, but no reproducible results were obtained by employing this system, and the system was not able to demonstrate the ability to separate the proteins. Further, the fabrication methodology used to produce this microfabricated electrophoretic device was elaborate, and the lithography procedure that was employed ultimately determined the minimum pore size that could be attained using this approach, thus suggesting that nanoporous membranes, with pore sizes in the order of tens of nm which are needed for some biological separations, could not be produced with conventional microfabrication techniques.
Other researchers have utilized a method of integrating carbon nanotube growth and traditional microfabrication technology to create a “nanotube mesh” inside microchannels [7], which was used for filtering fluorescent beads. The beads, which were about 200 nm in diameter, were released and collected by applying a back pressure to the mesh. However, no electrokinetic separation of molecules could be accomplished by this method. In fabricating the microchannel, the silicon substrate that was used to create the microchannel was bonded to a glass coverslip by anodic bonding, and thus, because of the use of a semiconducting material, electrophoretic separation could not be performed. Also, since fluid flow was pressure driven, separation was completely based on analyte size. Analytes that require charge-based separation simply could not be separated by this technique. Finally, because that microchannel was etched in silicon, a semiconductor, the material rendered the device incompatible with electrochemical detection, which is one of the preferred detection methodologies in micro total analytical systems.
Accordingly, there remains a need in the art for an improved microfluidic device and method of using the same that can effectively be used to separate molecules electrokinetically, including separation by electrophoresis and electroosmosis. In particular, there remains a need in the art for a carbon nanotube-based microfluidic device that is capable of electrokinetically separating multiple biochemical analytes, including proteins and nucleic acids, in a sample of interest.